Hepatitis B virus (HBV) is an enveloped dsDNA virus with a ssRNA intermediate form. Correct assembly of the icosahedral core of HBV is required for replication; reverse transcription of the RNA pre-genome takes place within the completed capsid, the protein shell of the core. Current anti-HBV therapeutics are interferon and nucleoside analog inhibitors of HBV reverse transcriptase (Pol). Both approaches have limited efficacy and can be expensive. Like any monotherapy, Pol inhibitors select for resistant mutants; some of these mutations escape the vaccine because Pol and surface protein genes overlap. We advocate capsid assembly as a complementary target for antiviral therapy. Assembly is critical to HBV replication and is unique to the virus (there are no cellular homologs to capsid protein). The HBV capsid is constructed from 120 protein dimers. In vitro, assembly can be induced by a number of small molecules and salts. We have found that HBV assembly is allosterically regulated: capsid protein has assembly-active and -inactive conformations. Consequently, small molecules that favor the inactive form will inhibit assembly; molecules that favor the active form will accelerate the process. We have found that some molecules misdirect capsid assembly, presumably by distorting the dimer geometry, giving rise to the rapid production of misshapen and nonfunctional structures. At this time, we have a small selection of molecules in each category. In this proposal we describe a strategy for developing assembly-directed antivirals. Starting with the pre-existing leads, we will investigate the physical and structural basis for altered assembly by the most active molecules. Using this information, we will then take advantage of recent advances in the quantification of assembly to screen candidate small-molecule libraries for enhanced activity in assembly acceleration, inhibition, and misdirection. The effects of these molecules will also be tested in cultured cells that express HBV. More than 350 million individuals suffer from chronic infection with hepatitis B virus (HBV), including more than 1.25 million Americans. Worldwide, HBV will contribute to 1 million deaths this year. Current antiviral strategies focus on virus enzymes. We propose a new strategy for developing antiviral molecules that target virus assembly.